PROJECT 2: PROTEIN TURNOVER DYNAMICS SUMMARY Protein abundance levels are controlled through regulatory processes that govern protein synthesis and degradation. Although translational control is now a widely appreciated mechanism for regulating gene expression and proteome remodeling, a systems-level relationship between translational regulation and cellular physiology remains largely unexplored. The overarching objective of this project is to discover the mechanisms that lead to alterations in proteome flux and predict their responses to dynamic changes in the environment. This objective will be pursued through three aims. First, we will investigate how the availability of key components of the translational apparatus, such as ribosomes, tRNAs and initiation factors, are balanced with the transcriptome and proteome composition depending on a specific static carbon source. This will allow us to develop detailed mathematical models that link global translation, transcription and cell physiology. The predictions of the models can be tested by artificially perturbing the transcription-translation balance. Second, we will study the global feedback mechanisms by which the cell adapts its translational capacity to shifts in the carbon source experimentally, while in parallel extending our mathematical models to integrate the regulatory mechanisms linking carbon source and growth rate with a systems-view of the translation system. We will investigate, using experiments and mathematical models, how differences in the adaptation time of the various components of the translational machinery, proteome and mRNA composition to a fluctuating carbon source affect the cell's translational capacity, and hence, how the cell's global feedback mechanisms respond to dynamic and stochastic changes in the carbon source. Finally, we will build a quantitative mathematical model of proteome flux using a combination of quantitative proteomics and mathematical modeling. We will quantify key parameters of proteome flux including protein translation rate, protein degradation rate, mRNA abundance, and protein abundance. This will be done in both rapidly cycling cells and non-dividing neurons to determine how proteome flux is re-wired in post-mitotic cells. We will use serum starvation as a means to limit nutrients and measure alterations in proteome flux upon nutrient withdrawal and replenishment. We will also investigate dynamics in proteome flux upon mTOR inhibition as a pharmacological means to mimic nutrient deprivation. This will allow for deterministic modeling of how proteome resource allocation is altered, in two divergent cell types, upon nutrient limitation.